1. Field of the Invention
This invention, in one aspect, is directed to methods for recovering Shiga-like toxins (SLT) from a sample under physiologically acceptable conditions using an affinity ligand covalently attached to a solid support. The use of such a covalently attached affinity ligand enhances the purity of the product and these methods employ mildly basic conditions to effect elution of the SLT from the affinity support thereby avoiding the use of acidic conditions and, in particular, harsh acidic conditions.
In another aspect, the SLTs recovered in this invention are inactivated to provide for an immunoprotective vaccine.
2. References
The following references and patents are cited in this application as superscript numbers:
1. Boulanger et al., Universal Method for the Facile Production of Glycolipid/Lipid Matrices for the Affinity Purification of Binding Ligands, Analytical Biochem. 217:1-6 (1994). PA1 2. Armstrong et al., Investigation of Shiga-like Toxin Binding to Chemically Synthesized Oligosaccharide Sequences, J. Infect. Dis. 164:1160-1167 (1991). PA1 3 Pozsgay et al., Purification of Subunit B of Shiga Toxin Using a Synthetic Trisaccharide-Based Affinity Matrix, Bioconj. Chem. 7:45-55 (1996). PA1 4 Donohue-Rolfe et al., Purification of Shiga Toxin and Shiga-Like Toxins I and II by Receptor Analog Affinity Chromatography with Immobilized P1 Glycoprotein and Production of Cross-Reactive Monoclonal Antibodies, Infect. Immun. 57:3888-3893 (1989). PA1 5 Brown et al., Digalactosyl-Containing Glycolipids as Cell Surface Receptors for Shiga Toxin of Shigella dysenteriae 1 and Related Cytotoxins of Escherichia coli, Rev. Infect. Dis. 13(Suppl 4):S298-303 (1991). PA1 6 Ryd et al., Purification of Shiga toxin by .alpha.-D-galactose-(14)-.alpha.-D-galactose-(1-4)-.alpha.-D-glucose-(1-) receptor ligand-based chromatography, FEBS Letters 2:320-322 (1989). PA1 7 Acheson et al., One step High Yield Affinity Purification of Shiga-Like Toxin II Variants and Quantitation using Enzyme Linked Immunosorbent Assays, Microb. Pathog. 14:57-66 (1993). PA1 8. Donohue-Rolfe et al., Shiga Toxin: Purification, Structure, and Function, Rev. Infect. Dis. 13(Suppl 4):S293-7 (1991). PA1 9. Waddell et al., Induction of Verotoxin Sensitivity in Receptor-Deficient Cell Lines Using the Receptor Gtycolipid Globotriosylceramide, Proc. Natl. Acad. Sci. 87:7898-7901 (1990). PA1 10 Acheson et al., Expression and Purification of Shiga-Like Toxin II B Subunits, Infect. Immun. 63:301-308 (1995). PA1 11. O'Brien et al., Purification of Shigella Dysenteriae 1 (Shiga)-Like Toxin From Escherichia coli O157:H7 Strain Associated with Haemorrhagic Colitis, Lancet Sep. 3, 1983, page 573. PA1 12 Calderwood et al., A System for Production and Rapid Purification of Large Amounts of the Shiga Toxin/Shiga-Like Toxin I B Subunit, Infect. Immun. 58:2977-2982 (1990). PA1 13 Acheson et al., Enzyme-Linked Immunosorbent Assay for Shiga Toxin and Shiga-like Toxin II Using P1 Glycoprotein from Hydatid Cysts, J. Infect. Dis. 161:134-137 (1990). PA1 14 Armstrong, et al., Method of Removing Shiga-Like Toxins From Biological Samples, U.S. Pat. No. 5,620,858, issued Apr. 17, 1997. PA1 15 Rafter, et al., U.S. patent application Ser. No. 08/669,004, filed Jun. 21, 1996, TREATMENT OF BACTERIAL DYSENTERY PA1 16 Lemieux, R.U., et al., The properties of a `synthetic` antigen related to the blood-group Lewis A, J. Am. Chem. Soc., 97:4076-83 (1975). PA1 17 Ekborg, G., et al., Synthesis of Three Disaccharides for the Preparation of Immunogens bearing Immunodeterminants Known to Occur on Glycoproteins, Carbohydrate Research, 110: 55-67 (1982). PA1 18 Dahmen, J., et al., 2-Bromoethyl glycosides: applications in the synthesis of spacer-arm glycosides, Carbohydrate Research, 118: 292-301 (1983). PA1 19 Rana, S. S., et al., Synthesis of Phenyl 2-Acetamido-2-Deoxy-3-O-of-L-Fucopyranosyl-O-D-Glucopyranoside and Related Compounds, Carbohydrate Research, 91:149-157 (1981). PA1 20 Amvam-Zollo, P., et al., Streptococcus pneumoniae Type XIV Polysaccharide: Synthesis of a Repeating Branched Tetrasaccharide with Dioxa-Type Spacer-Arms, Carbohydrate Research, 150: 199-212 (1986). PA1 21 Paulsen, H., Synthese von oligosaccharid-determination mit amid-spacer vom typ des T-antigens, Carbohydr. Res., 104:195-219 (1982). PA1 22 Chernyak, A. Y., et al., A New Type of Carbohydrate-Containing Synthetic Antigen: Synthesis of Carbohydrate-Containing Polyacrylamide Copolymers having the Specificity of 0:3 and 0:4 Factors of Salmonella, Carbohydrate Research, 128:269-282 (1984). PA1 23 Fernandez-Santana, V., et al., Glycosides of Monoalkyl Diethylene Glycol. A New type of Spacer group for Synthetic Oligosaccharides, J. Carbohydrate Chemistry, 8(3):531-537 (1989). PA1 24 Lee, R. T., et al., Synthesis of 3-(2-Aminoethylthio) PropylGlycosides, Carbohydrate Research, 37:193-201 (1974). PA1 25 Lemieux, R. U., et al., Glycoside-Ether-Ester Compounds, U.S. Pat. No. 4,137,401, issued Jan. 30, 1979. PA1 26 Lemieux, R. U., et al., Artificial Oligosaccharide Antigenic Determinants, U.S. Pat. No. 4,238,473, issued Dec. 9, 1980. PA1 27 Lemieux, R. U., et al., Synthesis of 2-Amino-2-Deoxyglycoses and 2-Amino-2-Deoxyglycosides from glycals, U.S. Pat. No. 4,362,720, issued Dec. 7, 1982. PA1 28 Dahmen, J., et al., Synthesis of space arm, lipid, and ethyl gtycosides of the trisaccharide portion .alpha.-D-Gal-(1-4)-g-DGal(1-4)-O-D-Glc! of the blood group p.sup.k antigen: preparation of neoglycoproteins, Carbohydrate Research, 127: 15-25 (1984). PA1 29 Garegg, P. J., et al., A Synthesis of 8-Methoxycarbonyloctyl-1-yl O-a-D-Galactopyranosyl-(1-3)-O-O-D-Galactopyranosyl(1-4)-2-Acetamido-2-Deo xy-D-Glucopyranoside, Carbohy. Res., 136: 207-213 (1985). PA1 30 Rappuoli, R., Toxin Inactivation and Antigen Stabilization: Two Different Uses of Formaldehyde, Vaccine, 12:579-581 (1994). PA1 i) contacting said sample with an affinity support having an affinity ligand comprising the disaccharide subunit .alpha.Gal(1.fwdarw.4).beta.Gal covalently linked to the affinity support through a compatible linker arm to form a SLT-affinity support complex; PA1 ii) separating the SLT-affinity support complex from the sample; PA1 iii) recovering free SLT from the complex under basic nondenaturing conditions; PA1 wherein the purified SLT is essentially free of glycolipids. PA1 i) contacting said sample with an inert solid affinity support having a disaccharide subunit .alpha.Gal(1.fwdarw.4).beta.Gal covalently linked to the affinity support through a non-peptidyl compatible linker arm to form a SLT-I/affinity support complex; PA1 ii) separating the SLT-I/affinity support complex from the sample; PA1 iii) recovering free SLT-I from the complex by contacting the complex with an aqueous solution having a pH of from about 8 to 11 to provide SLT-I in the aqueous solution and the affinity support; PA1 iv) separating the aqueous solution from the affinity support. PA1 i) contacting said sample with an affinity support having an affinity ligand comprising the disaccharide subunit .alpha.Gal(1.fwdarw.4),.beta.Gal covalently linked to the affinity support through a non-peptidyl compatible linker arm to form a SLT-II/affinity support complex; PA1 ii) separating the SLT-II/affrnity support complex from the sample; PA1 iii) recovering free SLT-ll from the complex by contacting the complex with an aqueous basic solution of urea having a urea molarity of from about 0.5 to 3 M to provide SLT-II in the aqueous solution and the affinity support; PA1 iv) separating the aqueous solution from the affinity support.
All of the above references are herein incorporated by reference in their entirety to the same extent as if each individual reference was specifically and individually indicated to be incorporated herein by reference in its entirety.
3. State of the Art
Shigella dysenteriae type 1, is the Shigella serotype responsible for the most severe cases of bacillary dysentery. This bacteria produces a protein, Shiga toxin, that possesses potent neurotoxic, cytotoxic and enterotoxic effects which are well understood in the art and is the causative agent in shigellosis.
Escherichia coli is an indigenous member of the intestinal tract of humans and animals where it facilitates digestion. Enterovirulent E. coli organisms, however, differ from the normal E. coli residents of the intestinal tract because of their ability to invade the intestinal mucosa and to produce enterotoxins. Certain pathogenic strains of E. coli elaborate a toxin that is cytotoxic for African green monkey (Vero) cells. Hence, the term "Verotoxin" was introduced to describe this cytotoxic activity.
Verotoxins from different E. coli strains constitute a family of structurally and functionally related cytotoxins, the prototype of which is Shiga toxin. Thus, the term Shiga-like toxin (SLT) is synonymous with Verotoxin. Shiga-like toxins are proteins secreted by certain pathogenic strains of E. coli and are the causative agents of numerous disease conditions such as hemorrhagic colitis, hemolytic uremic syndrome and the like.
Different antigenically distinct SLTs have been described including SLT-I and SLT-II. SLT-I is nearly identical to Shiga toxin. SLT-II, including known variants of SLT-II, is related but is not neutralized by anti-Shiga toxin serum. Both toxins are comprised of multiple copies of a B subunit and a single A subunit. The B subunit is approximately 7.5 kDa and is associated with receptor binding. The A subunit is approximately 35 kDa and is responsible for the catalytic inhibition of protein synthesis. SLT-I and SLT-II are 60% homologous overall and 70% homologous in the B subunit.
SLT-producing E. coli belong to several different serotypes but all have in common the ability to secrete one or more SLTs. In North America, the 0157:H7 E. coli serotype is isolated from 95% of cases of SLT mediated infections whereas, in other locations, different enterohemorrhagic E. coli serotypes predominate. Serotype 0157:H7 E. coli can readily be identified in the clinical laboratory because of its inability to utilize sorbitol as a carbon source. However, clinical laboratories that rely only on sorbitol fermentation to test for SLT mediated infections will fail to identify such infections arising from non-0157:H7 E. coli serotypes. This, of course, is a major concern in those regions where non-0157:H7 serotypes predominate. Accordingly, clinical diagnosis of SLT mediated infections in a patient by assaying only for the presence of enterohemorrhagic 0157:H7 E. coli serotypes is not advised.
Another diagnostic method is the detection of SLTs in the stools of patients suspected of being infected with enterohemorrhagic E. coli. Diagnostic kits used in the detection of SLTs are now commercially available but, nevertheless, these tests require a purified source of SLT, preferably SLT I and SLT II, to serve as a positive control. Moreover, with increased recognition of this disease condition the need for and usage of these diagnostic kits will continue to increase. Thus, a facile, efficient method of recovering SLTs from a sample would be desirable.
In addition to diagnostic utility, methods for providing large quantities of purified SLTs are also required for use in prophylactic treatment regimens for patients, particularly patients with weakened immune systems, wherein inactivated forms of the SLTs could be used as an immunoprotective vaccine.
Specifically, enterohemorrhagic E. coli infections are treated clinically as self-limiting because antibiotics are of little therapeutic value. The failure of antibiotic therapy may relate to the central role of SLT in the disease, and antibiotics are not directed at reducing the activity of these toxins. Accordingly, alternative methods of therapeutically treating SLT mediated infections, including hemolytic uremic syndrome (HUS), have been proposed including the oral ingestion of an affinity ligand to the SLT which ligand is covalently attached to a solid inert support through a non-peptidyl linker arm..sup.14 In such treatment, the affinity ligand complexes with the toxin in vivo and is subsequently eliminated as part of the patient's stool thereby lowering toxin levels in the infected individual.
In such treatment regimens, it has been reported, however, that the incidence of HUS is reduced by the administration of this affinity ligand during a critical period after onset of the disease..sup.15 While such time critical treatment regimens would, of course, reduce the incidence of HUS, the difficulty in diagnosis of the SLT mediated disease conditions coupled with the possibility that the infected individual would not present himself/herself to a physician during this critical time period, suggests that prophylactic methods to prevent enterohemorrhagic E. coil infection are desirable, particularly in individuals susceptible to such infections.
As it relates to this last aspect, it is a fundamental that a major defense mechanism of humans and animals against infection by pathogenic organisms, such as SLT-producing E. coli, is their ability to produce antibodies that bind to the pathogens and their toxins, inactivating them or preparing them for destruction by specialized cells in the body. In the very young, i.e., infants, an undeveloped immune system may not provide adequate protection against such infections. In elderly patients, an incompetent immune system may likewise fail to provide protection. Accordingly, any person with a compromised immune system could suffer a lethal infection upon first exposure to enterohemorrhagic E. coli infection if their immune system was not primed. One method for so priming such persons would be to administer an immunoprotective vaccine to that person which vaccine would prophylactically act to prevent the occurrence of this disease.
Such vaccines would, of course, require purification of SLTs which are subsequently inactivated. Various methods have been heretofore disclosed for isolating SLTs. These methods, however, are in one manner or another not preferred for preparing large quantities of purified SLTs for use in a vaccine. For example, receptor analog affinity chromatography with a glycoprotein present in hydatid cyst fluid has been utiized..sup.4,7,10,13 This glycoprotein possesses a trisaccharide, .alpha.Gal(1.fwdarw.4).beta.Gal(1-4)GlcNAc, that is identical to the erythrocyte P1 glycolipid. However, safety concerns regarding possible contamination of the isolated SLTs would preclude the use of these recovered SLTs in vaccine preparations.
The glycolipid Gb.sub.3 (.alpha.Gal(1.fwdarw.4).beta.Gal(1.fwdarw.4).beta.Glc-ceramide) has also been used to bind and remove SLTs from a sample. In Boulanger.sup.1, the Gb.sub.3 was adsorbed non-covalently onto Celite. The lack of a covalent linkage between the glycolipid and the affinity support would, in principle, allow the glycolipid to leach from the affinity support. Upon elution of the SLTs one cannot ensure that some of the glycolipid has leached from the affinity support into the SLT. Thus, contamination of the SLTs by small amounts of Gb.sub.3 is possible and such contamination would preclude its use in the preparation of a vaccine.
Others have used glycoconjugates or glycolipids containing the disaccharide sequence .alpha.Gal(1.fwdarw.4).beta.Gal covalently linked to a solid support to bind the SLTs. Subsequent elution from these substrates has required harsh and/or denaturing conditions. For example, guanidine HCl.sup.3,6, 10% SDS in boiling water.sup.2, MgCl.sub.2.sup.4,7,8,10,12 all have been used. The protein thus recovered may lose antigenic epitopes, be less immunogenic and, consequently, provide an inferior vaccine.
From the above it is apparent that a need for a rapid, inexpensive method of recovering shiga-like toxins is desirable. Further, a safe, immunoprotective vaccine is desirable.